The objective of the proposed research is to develop a novel in vivo microscopic imaging technology, confocal photoacoustic microscopy (PAM). PAM can image intact biological tissues at high resolution with optical absorption contrast, which is sensitive to physiological parameters such as the total hemoglobin concentration and the oxygenation of hemoglobin. PAM has potentially broad applications in both small-animal research and clinical practice. For example, PAM can potentially be applied to (1) the animal study of angiogenesis, brain function, drug discovery, and molecular cell biology, (2) the clinical diagnosis of skin cancer and cervical cancer, and (3) the intra-operative demarcation of brain cancer. In addition, PAM can be adapted for high-resolution molecular imaging. Although optical contrast is high, existing high-resolution optical imaging modalities-including confocal microscopy, two-photon microscopy, and optical coherence tomography (OCT)-cannot penetrate more than ~1 mm in scattering biological tissue. This depth has been a fundamental limit of existing high-resolution optical imaging modalities for decades owing to strong optical scattering. Furthermore, the current modalities are not directly sensitive to optical absorption. PAM combines optical contrast with ultrasonic resolution;it breaks through the 1-mm depth limit while providing high spatial resolution with a depth-to-resolution ratio greater than 100, as demonstrated by our recent work published in Nature Biotechnology. Our existing 50-MHz system can provide a maximum imaging depth of 3 mm, an axial resolution of 15 5m, and a lateral resolution of 45 5m. No other optical technology can image blood vessels and functions at this resolution and depth. More importantly, the maximum imaging depth and the spatial resolution of PAM can be scaled with the ultrasonic parameters. Furthermore, PAM is free of speckle artifacts, which plague OCT and ultrasonography. Potential applications were highlighted by Nature Reviews Drug Discovery and Nature Reviews Molecular Cell Biology. In the proposed technology-driven research, we will focus on pushing the limits of PAM with the following specific aims: 1. To develop a higher-frame-rate system 2. To develop a hand-held scanning probe 3. To develop a higher-resolution system 4. To develop a deeper-penetration system 5. To adapt a commercial ultrasound imaging system. PUBLIC HEALTH RELEVANCE: Imaging technologies have enabled numerous discoveries in biomedicine and provided early diagnosis of disease. Functional imaging that detects not only tissue structure but also tissue function will make even greater impact in biomedicine. The proposed photoacoustic imaging technology represents a novel high-resolution functional imaging modality.